Adipocyte HSL is required for maintaining circulating vitamin A and RBP4 levels during fasting

Vitamin A (retinol) is distributed via the blood bound to its specific carrier protein, retinol-binding protein 4 (RBP4). Retinol-loaded RBP4 is secreted into the circulation exclusively from hepatocytes, thereby mobilizing hepatic retinoid stores that represent the major vitamin A reserves in the body. The relevance of extrahepatic retinoid stores for circulating retinol and RBP4 levels that are usually kept within narrow physiological limits is unknown. Here, we show that fasting affects retinoid mobilization in a tissue-specific manner, and that hormone-sensitive lipase (HSL) in adipose tissue is required to maintain serum concentrations of retinol and RBP4 during fasting in mice. We found that extracellular retinol-free apo-RBP4 induces retinol release by adipocytes in an HSL-dependent manner. Consistently, global or adipocyte-specific HSL deficiency leads to an accumulation of retinoids in adipose tissue and a drop of serum retinol and RBP4 during fasting, which affects retinoid-responsive gene expression in eye and kidney and lowers renal retinoid content. These findings establish a novel crosstalk between liver and adipose tissue retinoid stores for the maintenance of systemic vitamin A homeostasis during fasting.


Figure
Figure EV2.RBP4 accumulation and secretion in primary hepatocytes is regulated by cAMP signaling but not by all-trans retinoic acid (atRA).
(A) Primary hepatocytes were incubated with increasing concentrations of atRA and RBP4 protein in cell lysates and media analyzed by immunoblotting, RAN protein and Ponceau membrane staining served as loading controls, respectively.(B) Densitometric analysis of blots shown in (A).(C) Hepatocytes were incubated with 0.5 mM of 8-Br-cAMP for 24 h and RBP4 protein in cell lysates and media analyzed by immunoblotting, RAN protein and Ponceau membrane staining served as loading controls, respectively.10 µM of the proteasome inhibitor MG132 was added to vehicle and 8-Br-cAMP-treated hepatocytes for the last 4 h before harvesting.(D) Densitometric analysis of blots shown in (C).Data information: Data are represented as individual data points of n = 3 for each condition and mean ± s.e.m. and *P < 0.05 vs. vehicle treatment using an unpaired two-tailed Student's t test (D).

Figure EV3 .
Figure EV3.Global Hsl knockout increases retinyl ester content in WAT but not liver and reduces expression of RBP4 in WAT.(A) HSL protein abundance in liver (left panel) and perigonadal white adipose tissue (pgWAT) (right panel) of wild-type (wt) and Hsl knockout (ko) mice was determined by immunoblotting.GAPDH protein served as loading control.(B) Tissue retinol and retinyl esters in liver and pgWAT were analyzed by HPLC.Retinoids are shown as n/ µmol per total organ.(C) Wt and Hsl ko mice were fed and fasted as depicted and (D) abundance of RBP4 protein in livers was determined by immunoblotting.GAPDH served as loading control.(E) Densitometric analysis of blots shown in (D).(F) mRNA expression of Rbp4 and (G) that of canonical PPAR target genes in pgWAT was determined by qPCR.(H) Hepatic abundance of RBP4 was determined by immunoblotting, GAPDH served as loading control.(I) Densitometric analysis of the blots shown in (H).Data information: Data are represented as individual data points of n = 3, 3 (A), n = 6, 6 (B), n = 3, 3, 4, 4 (D, E), n = 6, 5, 6, 6 (F), n = 4, 4 (G), and n = 3, 3, 4, 4 (H, I) biological replicates and mean ± s.e.m., *P < 0.05 vs. wt mice using an unpaired two-tailed Student's t test (B, G) or a two-way ANOVA with Sidak's correction for multiple testing (E, F, I).

Figure EV4 .
Figure EV4.Global deletion of HSL does not impair hepatic retinol mobilization upon feeding Vitamin A-deficient diet (VAD).
(A) Mice of indicated genotype were fed normal chow or VAD and fasted for 16 h or not prior plasma and tissue collection as depicted.(B) Plasma retinol in ad libitum-fed mice on VAD for 9 weeks was determined by HPLC.(C) Plasma retinol in fasted mice on VAD for 9 weeks was determined by HPLC.(D) Retinol (left panel) and retinyl ester content (right panel) of liver after feeding normal chow or VAD in fasted mice was determined by HPLC.Data information: Data are represented as individual data points of n = 5, 5 (B), n = 8, 6 (C), and n = 4 for each group (D) biological replicates and mean ± s.e.m., *P < 0.05 vs. wt mice using an unpaired two-tailed Student's t test (C).

Figure EV5 .
Figure EV5.Adipose tissue-specific Hsl knockout does not affect mRNA and protein expression of hepatic RBP4 but reduces RBP4 levels in WAT.(A) HSL protein abundance in liver (left panel) and perigonadal white adipose tissue (pgWAT, right panel) of ADIPOQ-Cre(−) and Cre(+) mice with floxed Hsl alleles was determined by immunoblotting.ACTB protein served as loading control.(B) ADIPOQ-Cre(-) and Cre(+) mice with floxed Hsl alleles were fasted as depicted and (C) hepatic mRNA expression of Rbp4 determined by qPCR.(D) Abundance of RBP4 protein in livers of mice described in (B) was determined by immunoblotting.ACTB served as loading control.(E) Densitometric analysis of blots shown in (D).(F) mRNA expression of Rbp4 and (G) that of canonical PPAR target genes in pgWAT of fasted mice was determined by qPCR.(H) Abundance of RBP4 in pgWAT of fasted mice was determined by immunoblotting, GAPDH served as loading control.(I) Densitometric analysis of the blots shown in (H).Data information: Data are represented as individual data points of n = 3, 3, 6, 6 (C), n = 3, 3, 4, 4 (E), n = 4, 3 (F), n = 5, 6 (G), n = 6, 5 (I) biological replicates and mean ± s.e.m., *P < 0.05 vs. Cre(−) mice using an unpaired two-tailed Student's t test (F, G, I).